Niu et al. BMC Medical Genetics(2020) 21:211
https://doi.org/10.1186/s12881-020-01148-1

CASE REPORTOpen AccessA nonsense variant in FBN1 caused
autosomal dominant Marfan syndrome in a
Chinese family: a case report
Yuping Niu1,2,3, Sexin Huang1,2,3, Zeyu Wang4, Peiwen Xu1,2,3, Lijuan Wang1,2,3, Jie Li1,2,3, Ming Gao1,2,3,
Xuan Gao1,2,3* and Yuan Gao1,2,3*

Abstract
Background: Marfan syndrome (MFS) is a common autosomal dominant inherited disease, and the occurrence rateis around 0.1–0.2‰. The causative variant of FNB1 gene accounts for approximately 70–80% of all MFS cases. In thisstudy, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene. This finding extended
the variant spectrum of the FBN1 gene and will provide a solution for patients to bear healthy offspring by
preimplantation genetic testing or prenatal diagnosis.
Case presentation: The patient was treated due to tachycardia during excitement in a hospital. Echocardiography
showed dilatation of the ascending aorta and main pulmonary artery, mitral regurgitation (mild), tricuspid
regurgitation (mild), and abnormal left ventricular filling. Electrocardiograph showed sinus rhythm. In addition,
flutters of shadows in front of his eyes and vitreous opacity were present in the patient. Genomic DNA wasextracted from peripheral blood samples from members of the family and 100 unrelated controls. Potential variants
were screened out by next-generation sequencing and confirmed by MLPA & Sanger sequencing. Real-time
fluorescence quantitative PCR (RT-qPCR) was performed to detect the relative mRNA quantitation in the patient. A
heterozygous nonsense variant c.3217G > T of the FBN1 gene, which resulted in p. Glu1073Term, was identified inboth patients. Only wild type bases were found in the cDNA sequence of the patient. Real-time fluorogenic
quantitative PCR results showed that the relative expression level of FBN1 cDNA in the patient was only about 21%
compared to that of normal individuals. This variant c.3217G > T of the FBN1 gene introduces a Stop codon in the
cb-EGF12 domain. We speculated that a premature translational-termination codon (PTC) was located in the mRNAand the target mRNA was disintegrated through a process known as nonsense-mediated mRNA decay (NMD),
which led to a significant decrease of the fibrillin-1 protein, eventually causing clinical symptoms in the patient.
Conclusions: In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene,which eventually led to Marfan syndrome in a Chinese family.
Keywords: Marfan syndrome, FBN1 gene, Nonsense variant, Nonsense-mediated mRNA decay* Correspondence: gaoxuan@sduivf.com; gaoyuan@sduivf.com
1
Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong
University, Jinan 250012, China
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Niu et al. BMC Medical Genetics(2020) 21:211

BackgroundMarfan syndrome, or MFS, is a common autosomal
dominant inherited connective tissue disorder with significant genetic diversity and variety in clinics. The incidence rate of MFS is around 0.1–0.2‰ [1] and about
0.1‰ in China [2]. MFS mainly devastates the skeletal,
cardiovascular, and ocular systems, but all connective
tissues around the body could potentially be affected [3].
MFS is primarily caused by a heterozygous variant of the
FBN1 gene located in 15q21.1. Currently, according to
the HGMD database, over 2200 different pathologicalvariants have been reported. The FBN1 gene is composed of 65 exons that code the protein fibrillin 1, a vital
component of connective tissues [4].
The FBN1 gene encodes fibrillin-1. Fibrillin-1 is a
350kD calcium binding glycoprotein with modular structure and forty seven EGF (epidermal-growth-factor)
structural domains of which forty three are cb-EGF (calcium binding) domains [5]. Each EGF site has six highly
conserved cysteine residues, which forms disulfide bonds
to maintain the 3D protein structure [6]. Six fibrillin-1
monomers combine to form a large complicated extracellular aggregate known as a “microfiber”, which is crucial to the integrity and stability of both elastic andinelastic tissues [4, 7]. A decrease in the number ofmicrofibers or the abrupt loss of microfiber function
would lead to interruption of the TGF-β signal [8], subsequently shutting down the pathway of fibrillin-1,
largely disturbing the role of the skeletal, cardiovascular,
and ocular systems.
In this study, 22 MFS relative genes were screened by
next-generation sequencing so as to seek out the target
variant in a MFS male patient, and a heterozygousc.3217G > T (p.Glu1073*) nonsense variant of the FBN1
gene was discovered. This variant resulted in the relativelower expression level of FBN1 in the patient than normal, which probably caused his MFS.
Case presentation
Patients and clinical presentation

Following the tenets of the Declaration of Helsinki, an
informed consent was signed for the study and the experimental protocol was approved by the Ethics Committee. On the premise of informed consent, theFig. 1 Pedigree chart of the patients

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peripheral blood of the pedigree members was collected
for testing, and the peripheral blood of 100 phenotypically normal people was collected as a control [from volunteers with a normal phenotype in our center].
An MFS family with three individuals in this study was
genetically tested in Fig. 1 (II9, II10, III5). The patient(III5, Fig. 1) was an adult and was diagnosed as MFS
with dilatation of the ascending aorta and main pulmonary artery, mitral regurgitation (mild), tricuspid regurgitation (mild), and abnormal left ventricular filling by
echocardiography (Fig. 2a). Meanwhile, electrocardiograph showed sinus rhythm, and vitreous opacity waspresent (Fig. 2b). The fingers and toes of the patient’s
mother (II9, Fig. 1) and aunt (II4, Fig. 1) had deformities,
though his father did not have a specific abnormal phenotype. The patient’s grandfather (I2, Fig. 1) and uncle (II1,
Fig. 1) are both highly myopic, and his grandfather (I2,
Fig. 1) and three cousins (III1, III2, III4, Fig. 1) died ofheart disease (they are all maternal relatives). In July 2018,
the patient underwent molecular genetic testing in ourcenter.
Genetic analysis

Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated
phenotypic normal controls using the Lab-Aid® 820 Nucleic Acid Extraction Mini Kit (Zeesan Biotech Co., Ltd.,
China). Twenty-two MFS relative genes were screened by
next-generation sequencing for variant seeking (NovaSeq
6000, Illumina, USA), and large fragment deletions or repeated variants of the FBN1 gene were detected by multiplex ligation-dependent probe amplification (MLPA)
following the protocol of the SALSA MLPA P065 Marfan
Syndrome-1 probemix and P066 Marfan Syndrome-2 probemix kit (MRC-Holland).
The candidate variants were confirmed and determined whether they co-segregated in this pedigree by
Sanger sequencing. Primers were designed using Primer3 online software (http://bioinfo.ut.ee/primer3/) andsynthesized by Invitrogen Biotechnology Co., Ltd. The
target variants PCR products were sequenced by Sanger
sequencing (BigDye® Terminator Cycle Sequencing Kit,
ABI 3730, America). Sequencing data was blasted online(https://blast.ncbi.nlm.nih.gov/). One hundred unrelated

Niu et al. BMC Medical Genetics(2020) 21:211

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Fig. 2 Clinical diagnosis of the proband. 2A: Ultrasonic cardiogram; 2B: Ocular ultrasound. Echocardiography showed dilatation of ascending aorta
and main pulmonary artery, mitral regurgitation (mild), tricuspid regurgitation (mild), and abnormal left ventricular filling (a). Ocular ultrasoundshowed vitreous opacity (b)control subjects were sequenced in the same way. Nextgeneration sequencing and Sanger sequencing data
(Fig. 3) indicated a c.3217G > T heterozygous variant in
the proband and his mother’s FBN1 gene (NM_000138.4) but not in the gene of the father and 100 unrelated controls. MLPA results showed that no fragment
loss or gain was found on the FBN1 gene exons (Fig. 4).
Total RNA was extracted from the blood samples
using Trizol (Takara Dalian, China), and cDNA synthesis
was performed using the PrimeScript™RT Master Mix(Perfect Real Time) (Takara Dalian, China). PCR and sequencing were performed in the same way as describedabove. cDNA Sanger sequencing results indicated that
only the wild type G base was detected at the c.3217
position of the proband’s FBN1 gene, and no T (variant)
was found (Fig. 5).
Real-time fluorescent quantitative PCR was performedby the LightCycler 480 SYBR Green I Master Kit (Roche,
Germany). The β-actin gene was used as a reference
gene. Detection data were analyzed using the 2-ΔΔCt
method. The ratios of gene expression values were normalized using that of β-actin. A melting curve analysis
was used to validate the reactions. The relative mRNA

expression of the FBN1 gene was normalized to the levelof β-actin mRNA. All statistical data were compiled
from ≥3 independent experiments. The following algorithm was used: ΔCt = CtFBN1-Ctβ-actin, ΔΔCt = ΔCt theproband -ΔCt the control. The difference is statistically different when 0.01 < P < 0.05, and extremely notable whenP ≤ 0.01. The melting curve of real-time fluorogenic
quantitative PCR reflected a high specificity in bothproducts of FBN1 and β-actin. Compared to control
groups, the relative expression level of the proband
FBN1 gene was only 21%, which was significantly lower
than normal (P ≤ 0.05) and also much lower than heterozygous prospective expression (Fig. 6).
The variation classification of this variant was analyzed
according to the American College of Medical Genetics
and Genomics (ACMG) standards and guidelines [9].
The heterozygous c.3217G > T variant discovered in thisstudy has not been documented in the HGMD databaseand is not reported in papers. Although this variant has
been reported in ClinVar with accession VCV000803093
and has been reported in dbSNP with rs137854478, thisvariant was also found in neither the Exome Aggregation
Consortium nor the 1000 Genomes Project population

Fig. 3 Genomic Sanger sequencing results of the patient, parents, and controls. a control group; b: the patient; c the patient’s mother; d thepatient’s father. The arrow indicates the c.3217 position of the FBN1 gene

Niu et al. BMC Medical Genetics(2020) 21:211

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Fig. 4 MLPA testing results of the patient. a MLPA P065 Marfan Syndrome-1 probemix; b MLPA P066 Marfan Syndrome-2 probemix

Fig. 5 cDNA Sanger Sequencing results of the patient and the control. a the control; b The patient. The arrow indicates the c.3217 position of
the FBN1 gene, only wild G base was found at the position in both the patient and control

Niu et al. BMC Medical Genetics(2020) 21:211

Fig. 6 Relative expression level of FBN1 gene of the proband is only
about 21% compared to the control group. β-actin was a reference
gene for analyses. The data represented the means of three
independent experiments done in duplicates. P-value was assessedusing a T-test. Significance p < 0.05 is indicated by (*); *p < 0.05
vs. control

database. According to an OMIM report, the patient’s
phenotype highly agrees with the symptoms of an autosomal dominant disorder known as Marfan syndromecaused by variants on the FBN1 gene located on
15q21.1. This variation was supposed to disrupt the roleof FBN1 by a named NMD (nonsense-mediated mRNA
decay) mechanism, which reduces gene production andaffects protein function. We classified the variant
as“pathogenic” (PVS1 + PM2 + PP4) according to theAmerican College of Medical Genetics and Genomics(ACMG) standards and guidelines.

Discussion and conclusions
Marfan syndrome is a hereditary connective tissue disease
with high variability. Amongst over thirty different symptoms, the most prominent ones are lesions on the skeletal,
cardiovascular, and ocular tissues [3]. In clinical cases,
ectasia of aorta and aortic dissection are the two most lethal and important phenomena of MFS [10]. MFS causedby the FNB1 variant accounts for approximately 70–80%
of all cases [11], while other cases may be caused by variants in potentially relative genes such as ACTA2, MYH11,
MYLK, SMAD3, TGFBR1, TGFBR2, etc.
The FBN1 gene, located on chromosome 15, q21.1,
encodes 2871 amino acids. Currently, over 2200 different
variations have been discovered, and most of them relating to MFS. There are two major types of influences that
FBN1 genetic variants can have on the structure offibrillin-1: (1) nonsense variants, creating truncatedPage 5 of 7

proteins; (2) missense variants, leading to abnormal
fibrillin-1 with mistakenly displaced amino acids [12]. In
some nonsense variants, mRNA containing prematuretranslational-termination codon (PTC) rarely generates
truncated proteins because they are disintegrated
through a process known as nonsense-mediated mRNA
decay (NMD) [13]. In cases of missense variants, many
displacements of amino acids influence components of
crucial residues, such as the cysteine residue on cb-EGF
structural domains. These displacements lead to possible
mis-folding of structural domains and cause retention of
the molecule in particular cases [14, 15]. Some other variants may affect the binding process between calcium andthe cb-EGF domain, lowering the calcium affinity levels of
the binding domain, altering the interaction patterns between cb-EGF domains in series with each other, and subsequently changing the rigidity of the molecule [16, 17].
In this research, we discovered that while the patient
showed symptoms of classical MFS, a c.3217G > T heterozygous variant occurred on the26th exon of the
FBN1 gene. This nonsense variant turned the 1073th
amino acid, a Glutamic acid, into a stop codon. In themeantime, only a wild type base was found in the cDNA
sequence of the patients and the target mRNA expression level was only 21% compared to the controls. Thus,
we speculated that the cell degraded mRNA through
NMD, which led to a significant decrease in the expression level of the fibrillin-1 protein, eventually causing
MFS [18, 19]. Tjeldhorn [20] et al. pointed out back in
1995 that nonsense or missense variants could cause alterations of mRNA expression, which agreed with our
results. The mRNA relative expression level of another
MFS patient (FBN1 c.4414 T > C: p.Cys1472Arg) in ourcenter tested by RT-PCR was 145.6% compared to thecontrol samples, once again confirming the possible
PTC mechanism.
On the other hand, a very similar variant of c.3217G >A, p.Glu1073Lys reported by Nijbroek [21], caused a severe symptom in newborn MFS. This variant affects thecb-EGD12 domain of the fibrillin-1 protein and each
EGF-like domain has six highly conserved cysteine residues, which form disulfide bonds to establish the tertiary
structure of the protein. The c.3217G > A: p.Glu1073Lys
variant causes the replacement of glutamic acid by lysine, which changes the electric charge of the amino acidfrom negative to positive in the anterior of first cysteinein the domain. And it is highly correlated with a decrease in the binding affinity between the cb-EGF12 domain and calcium [22]. “Dominant negative mutation” isa term for a pathogenic mechanism currently proposed
for many autosomal dominant diseases, including MFS[23, 24]. According to this model, a mutant gene product has the capacity to impair the function of the widetype protein produced by the normal allele. When

Niu et al. BMC Medical Genetics(2020) 21:211

considering a multimeric protein, interaction between
mutant monomer and the wild-type product can resultin the formation of abnormal multimer and hence thedisease phenotype [25]. This is why the variant of c. 3217
G > A caused a severe MFS symptom. The variant ofc.3217G > T found in this study was a nonsense variant,which produced a premature translational-termination
codon that reduces the stability of the variant transcript
and consequently reduces protein production from thevariant copy of the gene [26]. The production of the protein was decreased, but the protein produced by wild typegene was still there, giving rise to a mild MFS phenotype
by haploinsufficiency. The mechanisms and pathway affected by different variants at the same DNA position
would probably lead to entirely different phenotypes. Thishypothesis requires further studies to be verified.
In conclusion, we discovered a nonsense variant of the
FBN1 gene, c.3217G > T, p.Glu1037Ter, which is very liketo directly cause Marfan syndrome. This finding extended
the variant spectrum of FBN1 gene and will provide a solution for patients to bear healthy offspring by preimplantation genetic testing or prenatal diagnosis. This study isbased on data at the molecular level, and more experiments are needed to clarify the mechanism in the future.
Abbreviations
MFS: Marfan syndrome; PTC: premature translational-termination codon;
NMD: nonsense-mediated mRNA decay
Acknowledgements
The authors acknowledge and appreciate the generosity of their patientsand their relatives who participated in the study.
Authors’ contributions
YN was involved in the study design, data analysis, and manuscript drafting.
JL, PX, and SH did the clinical assessment and recruitment of the patientsand their family members. SH, LW and MG performed the experiments andcritically reviewed the manuscript. ZW, JL, and PX were involved in the study
design and critical evaluation of the manuscript. XG and YG conceived thestudy and wrote the manuscript. All authors have read and approved themanuscript.
Funding
The funders provided financial support.
The National Key Research and Development Program of China(2018YFC1004900; 2018YFC1003100), Key Research and Development
Program of Shandong Province (2017G006035), Natural Science Foundationof Shandong Province (ZR2018PH006, ZR2018MC014).
Availability of data and materials
All data generated or analyzed during this study are available from thecorresponding author upon reasonable request. The RNA sequencing data
has been uploaded to NCBI, and GenBank accession numbers are“BankIt2259986 Seq1 MN413940” and “BankIt2259986 Seq2 MN413941”.
Ethics approval and consent to participate
The study protocol was approved by the Ethics Committees of theInstitutional Review Board of Center for Reproductive Medicine of Shandong
University and all participants provided written informed consent.
Consent for publication
Written informed consent was obtained from all the participants for the
publication of this report and any accompanying images. A copy of thewritten consent is available for review by the Editor of this journal. All

Page 6 of 7

participants were over 18 years of age and had their written informed
consent for the publication of clinical details and/or clinical images.
Competing interests
The authors declare that they have no competing interests.
Author details
Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong
University, Jinan 250012, China. 2National Research Center for Assisted
Reproductive Technology and Reproductive Genetics, Shandong University,
Jinan 250012, China. 3Key laboratory of Reproductive Endocrinology of
Ministry of Education, Shandong University, Jinan 250012, China. 4Georgia
Institute of Technology, Atlanta, GA, USA.
1

Received: 6 May 2020 Accepted: 13 October 2020

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